Genetic Crosses

 A Monohybrid cross is a cross between two homozygous individuals for the study of a character. Monohybrid crosses the inheritance of a single character at a time. Through monohybrid cross law dominance can be determined.

                                                      TT x tt

                                                          ⇩

                          F1 generation        Tt( tall) x Tt(tall)

                                                           ⇩

Male/female gamete ➡   ⇩	T	t
T	TT (tall)	Tt (tall)
t	Tt (tall)	tt (short)

 According to the law of dominance of the two alleles of a gene, one is dominant over the other. The dominant gene expresses itself in one generation. As well in up to generation recessive genes completely mask recessive genes in the F1 generation. But in F2 generation also Expresses itself showing that the gene is not lost in the F1 generation. It is only expressed in homozygous conditions. Monohybrid cross can be in test cross where the individual of the F2 generation is crossed with a recessive parent this cross determines homozygosity or heterozygosity of the individual. If the F2 individual is homozygous dominant, the test cross ratio will be 3:1, if the F2 individual is heterozygous the test cross ratio will be 2:2.

                                                    Tt x tt 

                                                          ⇩             

Male/female gametes➡ ⇩	T	t
t	Tt Tall	tt short
t	Tt Tall	tt short

 

 Dihybrid cross is a cross to study two characters at a time. Dihybrid cross between a pea plant with it and a pea plant with green wrinkled seed shot up to a ratio of 9:3:3:1.  There is a parental type as well as a recombinant type of seeds. The parental type that is round yellow which were both dominant characters appeared in the 9/16 ratio. While the recombinant i.e. green round 3/16, wrinkled yellow 1/16. This show that the seed color and shape depend upon two different genes, which are inherited independently of each other in the next generation. All the possible four types of gametes are formed. Each type of gamete has a 25% possibility. This is called the law of independent segregation where genes segregate from each other independently. They combine in next-generation and result in the formation of parental type as well as recombinant individuals in the F2 generation.

Male/ female gamete	WG	Wg	wG	wg
WG	WWGG Yellow round	WWGg Yellow round	WwGG Yellow round	WwGg Yellow round
Wg	WWGg Yellow round	WWgg Green round	WwGg Yellow round	Wwgg Green round
wG	WwGG Yellow round	WwGg Yellow round	wwGG yellow wrinkled	wwGg yellow wrinkled
wg	WwGg Yellow round	Wwgg Green round	wwGg yellow wrinkled	wwgg Green wrinkled

 Complementary gene

 F2 generation ratio is 9:7 complementary genes. are those where both the gene are required for the expression of a character. As seen in the flower color of Lathyrus odoratus where two genes control the expression of flower color. When both the genes are present in dominant form only then purple flower color will appear. But if a single gene is present in the dominant form white flower color is formed. It may be understood from a two-step reaction where one gene controls the formation of the intermediate product and the intermediate product is converted into purple flower color only in presence of the product of the second gene. So, in absence of any of these genes the purple flower color is not formed.

Supplementary gene interaction

It is an interaction between two independent genes. Here two independent genes when present in the dominant form together give a different phenotype when these are present in the dominant form individually give a different phenotype.

As coat color in mice AB together gives agouti, ab gives albino, AA and B give black color give albino coat color.

Duplicate gene

It is the interaction between two different genes that have the same phenotypic effect. This interaction has F2 generation 15:1. Fruit shape of Capsella bursa pastoris (shepherded purse). Which has two types of fruit triangular and oval. The shape of the fruit is determined by two independent genes present on different chromosomes. The dominant form of both the genes gives triangular fruit shape and recessive genes give oval shape.

 

 Incomplete dominance

This is against the law of complete dominance where heterozygous individuals show intermediate complete dominance in incomplete dominance the genotypic ratio as well as phenotypic ratios are the same as incomplete dominance. It can be studied in flower color of mirabilis Jalapa homozygous dominant has red flower color heterozygous has pink flower color and homozygous recessive show white flower color.

GENE THERAPY

Gene therapy

 Gene therapy is the medical approach to correcting a gene to cure a disease or disorder. There is a number of diseases that are caused due to dysfunctional genes or due to the gain of function of a gene.  Genes express themselves by synthesizing proteins. If a protein is required for a metabolic process and this protein is non-functional due to defected gene, the metabolism is affected. Curing the protein is a process of re-establishing the metabolism. This can be done by curing the gene or transferring the correct gene into the organism.

 Gene therapy includes somatic gene therapy where genes are edited or transferred into somatic cells of the organism. This therapy cures the disease in an individual. But it is not inheritable as the gene is edited in somatic cells. This can be used for genes that are specific in their expression in the organism.  Somatic gene therapy includes gene transferring and gene editing.

SOMATIC CELL GENE THERAPY

Gene editing involves the specific technique of knocking out or knocking in the new gene. This uses crisper cas 9 Technology.

  The transfer of genes is used for somatic gene therapy. However, vectors can be used to deliver genes to a specific site. Even though intravenous gene transfer can be done for this virus vectors are used. These viruses will deliver the correct gene to the site of its requirement.

The embryonic gene is the gene therapy where embryonic stem cells are cured for defected genes.

  Gene editing may be used in embryonic gene therapy. Here the organism is cured in the embryonic stage only. Gene transfer can also be used in the embryonic cell stage also.

RNA EDITING

 The new organism that is born will have corrected gene function. However, this approach has broad applicability but it is not allowed on the ethical ground. The embryonic stem cell gene therapy may lead to human cloning and the formation of designer babies.

 Uses of gene therapy

 It utilizes the organism-specific approach to cure a disease. No chemical drugs or antibiotics are used in therapy to avoid their side effects.

 Gene therapy genetic disorders can be corrected for which rarely any curing approach is available to cancel can be cured through gene therapy without utilizing chemotherapy or radiotherapy which is destructive to human health. Spinal muscular dystrophy can be cured through gene therapy.

 Immunosuppressant diseases are also cured by gene therapy.

 Leukemia can be cured through gene therapy

 Crisper cas9 Technology

 Crisper cas 9 has a small guide RNA along with A nucleases the guide RNA has a specific sequence. This is called PAM this sequence binds to the targeted gene nucleus and then recognizes the site for cutting the DNA. It cut the DNA to either generate knockout where a gene is rendered dysfunctional or knock-in where the gene is rendered functional Crisper cas 9 Technology initiates the cellular mechanism of gene recombination it can be done through homologous change recombination or non-homologous gene recombination. Both of these mechanisms are well established in the cell and they can generate knockout or knock-in.

Fermentation technology: Ethanol, Lactic acid, and amylase production

ETHANOL PRODUCTION

Ethanol is an organic acid. It is widely used as a solvent. Ethanol is produced from carbohydrates (sucrose and starch). Ethanol is used as fuel.

Microorganisms producing ethanol 

Both bacteria and fungi produce ethanol. Bacteria used for ethanol production are Zymomonas mobilis. The yeasts used for ethanol production are Saccharomyces cerevisiae, and Kluyveromyces fragilis.  

Factors affecting ethanol production

Ethanol is inhibitory in higher concentrations. So, the microorganisms used in industrial production must be tolerant to the high concentrations of ethanol.

Pure sugar solution can increase the yield of ethanol. Osmotic tolerance of microorganisms is also important for ethanol production.

Organisms must have a high specific growth rate.

pH for ethanol production is 5-7.

The temperature for ethanol production is 30ºC.

PRODUCTION OF ETHANOL 

The process of ethanol production involves the following steps

1) Preparation of nutrient medium

2) Fermentation

3) Distillation of ethanol.

Preparation of medium

Substrates used for ethanol production are-

Starch-containing roots, tubers, or grains

Molasses

Wood waste 

Starch used in ethanol production has a low yield.

The substrate is heated to soften the substrate. It is liquefaction.

 It is enzymatically metabolized into sugars

Molasse is also used as a source of sugar for ethanol production.

Continuous culture is used to increase ethanol production.

BIOSYNTHESIS OF ETHANOL

Product recovery

Product recovery of ethanol involves two main steps.

1) Separation of biomass, which involves centrifugation.

2) Distillation of ethanol, this step of product recovery for ethanol production is the most energy demanding. It increases the cost of production of ethanol.

So, the cheap source of sucrose is beneficial for ethanol production. 

Lactic acid production

 Lactic acid was the first organic acid that was produced in 1880. Lactic acid can be produced through chemical methods or through fermentation. There is a competition between the chemical production of lactic acid and fermentation.

 Lactic acid production by the chemical method is cheaper. The production of lactic acid through fermentation should be from a cheaper feedstock.

 Making it more economical through fermentation technology, it is challenging for lactic acid production.

The organisms that produce lactic acid are classified as homofermentative organisms or Hetero fermentative organisms. The homofermentative organisms produce especially the lactic acid only. However, Heterofermentative organisms produce lactic acid as well as other acids.  

BIOSYNTHESIS OF LACTIC ACID

Bio-synthesis of lactic acid occurs from glucose when oxygen is insufficient.  As we know that glucose is metabolized to pyruvate through glycolysis. Pyruvate is a feeder molecule of the Kreb cycle, where it is oxidatively metabolized into carbon dioxide and water. However, when oxygen is limiting Pyruvate is converted into lactic acid by the enzyme lactate dehydrogenase. Lactic acid is produced maybe Levo or dextrorotatory.

BIOSYNTHESIS OF LACTIC ACID

MICROORGANISMS USED FOR LACTIC ACID PRODUCTION

 Organisms those produced lactic acids are Lactobacillus brueckii and Lactobacillus leichmannii, Lactobacillus pentosus. These organisms are facultative anaerobes. Thus only reducing the presence of oxygen can induce the production of lactic acid.

 Lactic acid production does not require the complete exclusion of oxygen from the fermentation vessel.

The process of lactic acid production

 It requires the preparation of medium, inoculation, and extraction of product. Preparation of the medium requires calibrating the medium according to the needs of the organism. For bacterial culture, the medium contains containing 12 to 13% Glucose, Ammonium Phosphate 0.25%, Vitamin B, and calcium carbonate are used.

 The vessel size can be 25 to 120 m³.

The temperature of the culture is 45 to 50ºC. The maximum production of lactic acid occurs within 72 hours. So, the product is extracted after 72 hours. The higher concentration of lactic acid in the medium is poisonous to the bacteria. The concentration of lactic acid in the culture medium becomes up to 80 grams per liter per hour.

 

 

 

 Amylase Production

Amylase is a starch liquefaction enzyme. There are two types of amylase, α-amylase, and β-amylase. These enzymes are used in the sweetener industry for the conversion of starch into dextrins, maltose, or glucose. The conversion of starch into these products is called starch saccharification. 

 Both α-amylase and β-amylase are extracellular enzymes. These are produced by bacteria as well as fungi both the amylases attack on 1-4 α glycosidic bond of starch thus producing the product of different lengths. The products include dextrins which are oligosaccharides, maltose which are disaccharides, and glucose which are monosaccharides. 

All these products are sweet in taste so amylase found its application in the sweetener industry, baking industry, beverage industry, paper and pulp industry, and cloth industry. The enzymes that cause starch saccharification are α- amylase, β- amylase, and o- glucoamylase.  Other enzymes are isoamylase and x-pullulanase. These can metabolize amylopectin as well because they can attack 1-6 α- glycosidic bonds as well.

 The microorganisms that are used for amylase production

Bacterial species used in fermentation Technology for the production of amylase are

Bacillus subtilis,

 Bacillus cereus,

 Bacillus amyloliquefaciens,

 Pseudomonas and Thermofactor 

The thermophilic species used for amylase production is Thermobacter. 

It can be cultured at 53ºC.

 Fungi used in amylase production are

Aspergillus, 

Penicillium, 

Aspergillus oryzae,

 Mucor,

 Candida, 

and Rhizopus.

 Medium for amylase production by fungi has

 8% starch,

 1.2% NaNO3, 

0.1 MgSO4,

 2.0% Malt extract,

 0.05%KCl, 

0.003% FeSO4, 

0.08% Mg(NO3)2.

Medium used for amylase production by bacteria has

0.5% starch

0.56% NH4NO3

0.28% sodium citrate

0.13%KH2PO4

0.05% MgSO4.7H2O

0.01%CaCl2

0.05% peptone

0.2% yeast extract

Amylase is an extracellular enzyme. It is produced in the cytoplasm. It has a signal sequence that targets the enzyme to the cell membrane of the organism, where it is folded into three-dimensional structures which is the functional form of the enzyme. The enzyme is released into the extracellular matrix, where it converts starch into different sweeteners.

 Glucose present in the culture medium increases the growth of the bacteria but decreases amylase production.

 When nitrogen is a limiting factor in the medium, amylase production decreases. The maximum production occurs at 45ºC in 18 hours for bacterial species. Up to 3000 units/ml is produced at 27 ºC to 37 ºC.

 Amylase is a heat-tolerant protein that can tolerate up to 70°C temperature.

 Purification of amylase is done through protein purification Technology.

 

ANIMAL CLONING

 Cloning is making a copy of an organism. The animal clones produced have the same genetic makeup as that of the cloned organisms. 

Natural evidence of cloning is found in bacteria, yeast, and other single-cell organisms that reproduce vegetatively. 

In higher animals, evidence of cloning is not found. However, artificial animal cloning is possible. It was first successfully done by cloning Dolly, the sheep.  It was started in 1979. However, successful cloning of Dolly could be done only in 1996. 

Producing an animal of the same genetic makeup is called cloning. Clones can not be produced by the sexual reproductive process. Gene recombination occurs in sexual reproduction. Even the siblings are not clones. They differ from one another in their genetic makeup.

Animal cloning is somehow allowed in some countries, but human cloning despite different claims has not been done. Further, it is opposed on ethical grounds.

 Identical twins are clones, as they develop from the single fertilized cell, the zygote.

The technology of animal cloning: Somatic Cell Nuclear Transfer

The technology used for animal cloning is SCNT( Somatic Cell Nuclear Transfer).  It is the transfer of the somatic cell nucleus in the enucleated egg cell.

 Animals can not be cultured in vitro. The production of the animal clone from a Somatic cell requires stimulation of the somatic cell. This stimulation is done through the somatic nucleus transfer in enucleated egg cells.

Steps involved in animal cloning

Somatic cells from the skin of animals.

An unfertilized egg is isolated from the surrogate mother.

The egg is enucleated, through irradiation or suction of the nucleus using a micropipette.

The enucleated cell is fused with the somatic cell/nucleus, and an electric pulse is used to fuse these cells. Microinjection can also be used for SCNT.

The fused cell is then allowed to divide and develop in the embryo.

This is then transferred to the surrogate mother.

The embryo develops into a clone of the organism. 

Animal cloning 
( SCNT)

Uses of animal cloning

Animal cloning is used to 

 Improve cattle variety.

Increase milk and meat production.

Produce disease-free animals. 

Studying gene expression.

Study gene therapy.

Improve food production.

Limitations of animal cloning 

The clone produced is not a true copy of the animal.

As fur depends on the gene being expressed in an individual cell.

Aging occurs in the cells depending on telomer shrinking, cells cease to divide if the telomere shrinks excessively.

X- chromosome expression differs in somatic cells.

The success rate of animal cloning is very low. Dolly was produced after 276 unsuccessful attempts.

Disease-free animals are not produced through animal cloning as somatic cells become immunologically older.

Animals produced through genetic engineering are not allowed to be used as human food as their long-term effect on health is not known.